Lab report

Module 3: BROMELAIN EXTRACTION AND CHARACTERIZATION BY SDS-PAGE

The report should not exceed 11 pages

Format:
The report should be printed on an A4 sheet
Font: Times New Roman, size: 13
Line spacing: 1.5 lines
Margins: Top-3.5cm, bottom-3cm, left-3.5cm, right-2cm.
Page number is centered at the bottom of the page.
Every figure and table should have a caption. Figure captions are places below the figures.
Table captions are places above the tables.

1. Title Page (0.5 points)

o Descriptive title
o Course Name and Module
o Date Experiment Completed
o Group/Subgroup
o Name of student 1: ________________________ Student ID: ___________
o Name of student 2: ________________________ Student ID: ___________
o Name of student 3: ________________________ Student ID: ___________
o Representative student email:
2. Introduction (1.5 points)
o What is bromelain?
o What sources can you use to obtain bromelain? What source did you use to extract
bromelain in this module?
o What are the objectives of the experiments?
3. Materials and Methods (1 points) This section should include the flowcharts that
summarize the following experiments:
o Extraction of bromelain from pineapple
o Separation of bromelain by solvent precipitation
o Bradford assay
o Bromelain enzyme assay
o SDS-PAGE
4. Results (4 points):
Calculation
o How can you calculate the protein concentration?
o How can you calculate the bromelain activity?
o The formulas for calculating
 The total protein concentration in crude extract (I), bromelain extract
after solvent precipitation (II)
 Lab report
Module 3: BROMELAIN EXTRACTION AND CHARACTERIZATION BY SDS-PAGE

 The total enzyme activity in solution I and solution II

 The specific activity of an enzyme in solution I / II (i.e., the enzyme
activity per mg protein)
 The purification fold of bromelain after solvent precipitation
 The protein recovery rate (%)
 The bromelain activity recovery rate (%)

Data report and analysis

o Calculate the concentration of protein (µg/ml) in each of the five samples that
make up the standard curve and draw a calibration curve of Bradford assay.
Table 1. Bradford protein assay standard curve
Vol of BSA Vol of Protein Corrected
Test tubes 0.1 mg/mL deionized concentration OD595nm OD595nm
(mL) water (mL) (µg/mL) (∆OD595nm)
Blank 0 10
1 1 9
2 2 8
3 3 7
4 4 6
5 5 5

o Calculate the protein concentration in samples (I-II) using the standard curve.
Table 2. Determination of protein concentration in unknown samples

Protein
concentration Actual
OD595nm OD595nm OD595nm OD595nm Dilution
calculated protein
Samples (1) (2) (3) (Average) ∆OD595nm factor
based on the concentration
standard (µg/ml)
curve (µg/ml)
Solution
(I)
Solution
(II)
 Lab report
Module 3: BROMELAIN EXTRACTION AND CHARACTERIZATION BY SDS-PAGE

o Calculate the enzyme activity in samples (I-II)

Table 3. Determination of bromelain activity in unknown samples

Solution (I) Solution (II)

Samples
Blank Tyrosine Sample Blank Tyrosine Sample

OD620nm

OD620nm (Average)
∆OD620nm
Dilution factor
Amount of tyrosine (µg) in 0.5
mL solution used in “tyrosine”
batch
Amount of tyrosine (µg)
produced in “sample” batch
Bromelain activity (UI/ml)

o Show the results of protein and enzyme activity assays from the protein purification
in Table 4.
Table 4. Bromelain purification table

Bromelain
Total Total Specific Protein
Volume Purification activity
Step protein activity activity recovery
(ml) fold recovery
(mg) (UI) (UI/mg) (%)
(%)
Homogenate /
(crude extract)
(I)
Solvent
precipitation
(II)
 Lab report
Module 3: BROMELAIN EXTRACTION AND CHARACTERIZATION BY SDS-PAGE

o Show the result of SDS-PAGE: Label the protein ladder and the target protein,
and estimate the size of target protein based on the protein ladder
 Lab report
Module 3: BROMELAIN EXTRACTION AND CHARACTERIZATION BY SDS-PAGE

5. Discussion (2 points)
o Compare the protein concentration and bromelain activity of crude extract and
those of the sample obtained from solvent precipitation samples
o Compare the experimental results to theoretical results or results previously
reported by other research groups
o Estimate the size of the proteins in the samples based on the result of SDS-PAGE
6. Conclusion (0.5 points)
o The total protein concentration, enzyme activity, specific activity, purification fold,
protein recovery, activity recovery
o The estimated size of the proteins in the samples
7. Reference (0.5 points)